PROJECT SUMMARY/ABSTRACT The ionic milieu is an important source of signals for Mycobacterium tuberculosis (Mtb), key to its ability to adapt to the local environment. It further represents a facet whose inherent non-uniformity can serve to drive the marked heterogeneity in Mtb response and lesion outcome observed during infection, which is a critical impediment to efficient therapy. Indeed, novel fluorescent reporter Mtb strains have uncovered marked heterogeneity in the ionic environment (pH and chloride) and replication status of Mtb in vivo. However, how Mtb integrates information from multiple environmental cues is poorly understood, and there is a gap in knowledge of how drug treatment both affects, and is impacted by, the local environment experienced by individual Mtb in vivo. To address this critical gap in knowledge, Aim 1 of this project will define the mechanisms by which Mtb integrates response to disparate ionic signals. This will encompass (i) transcriptional studies of a recently identified master Mtb ionic signal regulator that affects bacterial response to pH, chloride, and potassium, and (ii) a screen for new master regulators, using a novel inducible transcription factor over-expression library in the background of a chloride and pH-responsive fluorescent reporter Mtb strain. Aim 2 will delineate how an integrated Mtb response to ionic cues affects infection heterogeneity and outcome, using deletion and inducible over-expression Mtb strains of critical master ionic signal regulators and a murine Mtb infection model that recapitulates lesion types observed in human infection. These studies are made possible through exploitation of unique fluorescent environmental and replication reporter Mtb strains, and an innovative imaging approach that enables single cell in vivo visualization and signal quantification. Finally, Aim 3 seeks to understand the relationship between local ionic environment and therapeutic modulation on Mtb replication and lesion properties in vivo. This will focus on two current drugs that are affected by, or influence, the ionic environment (pyrazinamide and clofazimine), as well as recently identified novel compounds that modulate Mtb response to chloride. This project is conceptually innovative in its focus on understanding mechanisms by which Mtb integrates information from multiple signals, particularly in the context of under-studied ionic cues. There is also innovation in the use of a novel integrated imaging approach to reveal Mtb response to environmental cues and drug treatment with single bacterium level resolution in vivo, while retaining spatial information from intact lesion and tissue architecture. These studies will illuminate critical environmental response integration nodes that represent novel therapeutic targets. Discoveries made will further build a model that drives the field beyond aggregate readouts of Mtb environmental adaptation and infection/treatment outcome, vital for achieving the mechanistic and molecular understanding of Mtb-host interactions in vivo required for the development of improved therapeutics.